Abstract

Dendritic cells (DCs) and monocyte-derived macrophages (MΦs) are key components of intestinal immunity. However, the lack of surface markers differentiating MΦs from DCs has hampered understanding of their respective functions. Here, we demonstrate that, using CD64 expression, MΦs can be distinguished from DCs in the intestine of both mice and humans. On that basis, we revisit the phenotype of intestinal DCs in the absence of contaminating MΦs and we delineate a developmental pathway in the healthy intestine that leads from newly extravasated Ly-6Chi monocytes to intestinal MΦs. We determine how inflammation impacts this pathway and show that T cell-mediated colitis is associated with massive recruitment of monocytes to the intestine and the mesenteric lymph node (MLN). There, these monocytes differentiate into inflammatory MΦs endowed with phagocytic activity and the ability to produce inducible nitric oxide synthase. In the MLNs, inflammatory MΦs are located in the T-cell zone and trigger the induction of proinflammatory T cells. Finally, T cell-mediated colitis develops irrespective of intestinal DC migration, an unexpected finding supporting an important role for MLN-resident inflammatory MΦs in the etiology of T cell-mediated colitis.

Supporting Information

Figure 1. Characterization of intestinal CD8⟨+-type and CD11b+-type DCs. Cells were prepared from the LP of the small and large intestine and from the MLN of Flt3L-deficient (A), B6 mice (A and B) or of CX3CR1-EGFP mice (C). After excluding NK cells, B cells, T cells, eosinophils and neutrophils, the remaining MHCII+ cells were divided into CD11chiCD64– Int-DCs and CD11c– to intCD64+ MΦs (see Fig. ). (A) CD11chiCD64– DCs Int-DCs are absent in the LP of Flt3L-deficient mice. (B) CD11chiCD64–-gated Int-DCs present in the intestinal LP were analyzed for CD24, CD11b, CD172⟨ and CD103 expression and categorized into CD24+CD11b–CD103+or– CD8⟨+-type DCs and CD11b+CD64–CD103+or– CD11b+-type DCs. The MLN contained a migratory counterpart for each of the Int-DC subsets. (C) Expression of CX3CR1 and F4/80 on CD24+CD11b– CD8⟨+-type DCs, CD11b+CD64– CD11b+-type DCs and CD11c– to intCD64+ MΦs from the specified tissues. In the MLN, Mig-DCs were identified as MHCIIhiCD11cinter-to-hiCCR7+ cells. The percentages of cells found in each of the specified gates are indicated. Data shown are representative of 3 independent experiments.

Figure 2. CD64 marks monocyte-derived MΦs in humans. (A) Human CD34+ cord-blood progenitor cells were infected with lentivirus coding for the GFP fluorescent protein and then injected into NSG mice. 8 weeks later, cells were prepared from the LP of the small and large intestine of humanized mice and GFP+ cells were analyzed by flow cytometry. After excluding B cells, T cells and NK cells, HLA-DR+ cells were analyzed for CD11c, BDCA1, BDCA3 and CD64 expression. (B) Cells were prepared from non-inflamed and inflamed sections of colon biopsy specimens obtained from patients with a history of IBD. After excluding B cells, NK cells and T cells, HLA-DR+ cells were analyzed for CD11c, CD14, BDCA1 and BDCA3 expression. Histograms correspond to CD64 expression on BDCA1+CD11c+, BDCA3+CD11c+, and CD14+CD11c+ cells. Note that human LP MΦs expressed higher levels of CD11c than those found on mouse LP MΦs. The percentages of cells found in each of the specified gates are indicated. Data in (A) are representative of 2 groups of 3 humanized mice. Due to the small amount of GFP+ cells recovered per mouse, the small and the large intestines were pooled for the analysis. Data in (B) are representative of biopsies from 4 individual patients.

Figure 5. Gating strategy used for the identification of CD64+ MΦs and of CD24+CD11b– and CD11b+CD64– DCs in the large intestine LP and the MLN of healthy and colitic mice. Cells were prepared from the LP of the large intestine (A) and the MLN (B) of steady-state B6 mice (B6 steady-state) and of Cd3e⊗5/⊗5 mice that had received CD4+Foxp3– T cells (IBD). After excluding NK cells, B cells, T cells, eosinophils and neutrophils, the remaining MHCII+ cells of the large intestine LP were analyzed for CD64, CD11c, CD24, CD11b and CCR7 expression and divided in the following subsets: MΦs (MHCII+CD11c– to intCD64+CD11b+), CD8⟨+-type (MHCII+CD11chiCD64–CD24+CD11b–) and CD11b+-type (MHCII+CD11chiCD64–CD11b+) Int-DCs. In the MLN, MΦs were identified as MHCII+CCR7–CD11c– to intCD64+CD11b+ cells and the MHCIIhiCCR7+CD64– Mig-DCs were subdivided into CD24+CD11b– CD8⟨+-type and CD11b+CD64– CD11b+-type Mig-DCs. Data are representative of at least 3 independent experiments.

Figure 6. CD64+ MΦs excel in phagocytosis and do not display a dendritic morphology. Cd3e⊗5/⊗5 mice were sacrificed 3 weeks after transfer of CD4+Foxp3– T cells. Light density cells were isolated from the MLN and FACS-sorted into MHCIIhiCD64–CD24+CD11b– CD8⟨+-type Mig-DCs, MHCIIhiCD64–CD11b+ CD11b+-type Mig-DCs and MHCIIhiCD64+CD11b+ CD64+ MΦs. The distinct subsets were then subjected to a microsphere phagocytosis assay and uptake of fluorescent microspheres was analyzed by confocal microscopy. Scale bars, 20 μm. Data are representative of 3 independent experiments.

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